Methods of detecting and determining an analyte which is contained in a sample, for example, a bio-component in the body fluid such as urine and blood, a trace amount of a substance existent in food, medicine, or natural environment, an industrial chemical substance, a trace amount of a substance contained in waste, or the like include ones for measuring a detectable substance such as a dyestuff formed by a reaction system in which the analyte is involved.
One of the methods is, for example, a method comprising subjecting hydrogen peroxide formed by the chemical reaction of the analyte and a reactive color coupler (dyestuff precursor) to an oxidation-reduction reaction in the presence of peroxidase (POD) and determining the formed dyestuff compound by colorimetry. This method is frequently used in clinical diagnosis and the like because of its simplicity. Another one of the methods is a method of measuring an analyte based on an electrochemical reaction for reducing/oxidizing with an electrode the oxidized/reduced form of an electron carrier (mediator) formed by an oxidation-reduction reaction between the electron carrier and the analyte caused by an enzyme or the like.
However, in the above conventional methods, as measurement sensitivity is not sufficiently high when the amount of the analyte is very small, a highly accurate measurement result cannot be obtained. Therefore, the development of a highly accurate measuring method having improved measurement sensitivity has been desired.
Further, since measurement takes long as a reaction takes time, or it takes time for a detection reaction to reach a termination, a rating method for carrying out quantitative determination from a reaction rate has such a problem that the accuracy of quantitative determination is low. To cope with this, to increase the reaction rate, the reaction system is heated, or the concentration of a reagent for the reaction is increased. However, in the method for heating the reaction system, a heat source is required for heating and analysis is thereby complicated. When the formed substance is thermally instable, detection is difficult and this means cannot be employed. The method for increasing the concentration of the reagent is not practical because it leads to a rise in the background of detection and an increase in the cost of analysis. There is also a method for adding a catalyst to increase the reaction rate. However, since there are many detection reactions for which preferred catalysts are unknown yet, this method is not practical as well. As described above, most of the conventional methods are still unsatisfactory and a novel method which enables quick measurement by increasing the reaction rate more simply has been eagerly desired.
When a reaction which forms a substance insoluble in a reaction solvent is included in the reaction system which forms a detectable substance, there is such inconvenience as enumerated below and called in question.    (1) In measurement in which optical detection is carried out using a liquid reagent, for example, in a batch type automatic biochemical test apparatus, when a dyestuff formed by a reaction is insoluble in a solvent, it separates out and adheres to the wall of a measurement cell to shield incident light or transmitted light or cause the pollution of a dispensing nozzle, and abnormality in absorption coefficient, diffusion or light shielding by agglomeration, thereby making measurement difficult.    (2) Similarly, in measurement in which optical detection is carried out using a liquid reagent, when an insoluble by-product is formed, it adheres to the wall of a measurement cell to shield incident light or transmitted light or cause the pollution of a dispensing nozzle, and diffusion or light shielding by agglomeration, thereby making measurement difficult.    (3) In measurement in which a dyestuff formed by a reaction caused by dropping onto or infiltrating a sample to be measured into a testing piece is optically detected, when the formed dyestuff is insoluble in a sample solvent, the dyestuff deposits on the substrate of the testing piece nonuniformly, or the agglomeration of the dyestuff occurs, thereby deteriorating measurement accuracy.    (4) In electrode measurement using a liquid reagent, for example, in a batch type automatic biochemical test apparatus, when an insoluble by-product is formed, the pollution of an electrode is caused by covering the surface of the electrode with the insoluble deposit, thereby reducing biochemical response and deteriorating measurement accuracy.
The difference between the words “insoluble” and “hardly soluble” indicates a difference in the degree of insolubility in a solvent. In the present invention, the word “insoluble” may be interchanged by “hardly soluble” in the following description.
Particularly, when the formed detectable substance is insoluble in a reaction solvent, reaction rate may be reduced, or measurement sensitivity may be lowered because the reaction system which forms the detectable substance is not uniform and the reaction does not proceed quickly in the prior art method. For example, in the reaction system using an enzyme, the reaction product may deposit near the enzyme or impede the reaction.
Therefore, a measuring method using a reaction which forms a by-product insoluble in a reaction solvent in which the reaction is carried out has rarely been employed. Accordingly, it has been necessary to select a reaction which does not form an insoluble product as a detection system or to develop a new detection reaction system by synthetic chemical means so that the product becomes soluble in a reaction solvent. However, these circumstances have limited a reaction system used. Meanwhile, much time and labor have been required for the research and development of a reaction system which forms only a soluble substance. Further, it has been necessary to add a surfactant for solubilizing, emulsifying or dispersing the product. However, the addition of a surfactant is disadvantageous from the view point of measurement cost and may produce an adverse effect such as interruption of a reaction. Therefore, it cannot be said that it is a perfect solution. Then, a novel method which solves this problem easily and enables measurement in the presence of an insoluble product has been ardently desired.
The method of measuring an analyte using a reaction system which forms hydrogen peroxide as described above is an important measuring method as there are many reactions which form hydrogen peroxide as a substance produced by oxidation. However, accurate measurement has not always been easy in the prior art methods for the following reasons. That is, in these measuring methods, the amount or concentration of a detectable substance such as a dyestuff compound must have a quantitative correlation with a specific substance such as hydrogen peroxide in some cases. However, an oxidation-reduction system in colorimetry is affected by the strong oxidizing activity of excessive hydrogen peroxide or the strong reducing activity of ascorbic acid or the like contained in a biological sample, and the above detectable substance such as a dyestuff compound decomposes, whereby a measurement error may be produced.
For example, in these measuring methods, when an excessive amount of hydrogen peroxide is temporarily formed from an analyte such as glucose by an oxidase such as glucose oxidase, a reaction between the formed dyestuff and hydrogen peroxide occurs in addition to a reaction between a dyestuff precursor and hydrogen peroxide. As a result, the formed dyestuff is decomposed by hydrogen peroxide as soon as it is formed and discolored.
When an enzyme such as peroxidase for producing active oxygen species such as a superoxide having high reactivity from hydrogen peroxide, or transition metal ions and a complex thereof exerting a similar function are existent in a sample, the active oxygen species react with the formed dyestuff, decomposes and discolors it. This interference has affected measurement adversely. When a reaction which forms a detectable substance such as a dyestuff is carried out while it is exposed to the air, the formed dyestuff may be oxidized by oxygen contained in the air or oxygen dissolved in a reaction solution, decomposed and discolored.
Therefore, various attempts have been made such as the research of a dyestuff precursor which provides a stable substance which is hardly decomposed and the addition of various stabilizers but these are still unsatisfactory.
Reducing substances such as ascorbic acid, uric acid and bilirubin contained in a biological sample have a great influence on an oxidation-reduction reaction. Particularly, how to measure an analyte accurately in the presence of ascorbic acid has been a significant theme in the field of clinical analysis for long time. Various interference suppression means such as selective decomposition with an enzyme, decomposition by the addition of periodic acid, oxidation decomposition with iron-ethylene diamine tetraacetate chelate, and selective separation with a semipermeable membrane have been tried in addition to the research of the above-described dyestuff precursor and the like (see Yoshihide Ohta, Yutaka Ogawa, Rinsho Kensa, 34 (4), 502–504 (1990); Japanese Patent Publication No. 1-41223 (1989); Japanese Patent Publication No. 2-4861 (1990); Japanese Patent Publication No. 4-18630 (1992); Japanese Patent Application Laid-open No. 5-95797 (1993); and Japanese Patent Application Laid-open No. 7-155196 (1995)).
There are further methods of measuring a specific analyte by forming a dyestuff (for example, an azo dyestuff) having quantitative relationship with the specific analyte by various known reactions other than the oxidation-reduction reaction (for example, condensation reactions such as an acid-base reaction and the coupling reaction of a diazonium salt, a complex forming reaction and the like) and optically determining the formed dyestuff. These methods are important measuring methods detailed in Bunseki Kagaku Binran (ed. by the Japan Society for Analytical Chemistry), for example. However, some of the thus formed dyestuffs may be an instable compound which is decomposed by oxygen in the atmosphere, an oxidizing or reducing substance contained in a sample, hydrogen ions or bases contained in the sample, light or the like. To measure this substance, for example, quick operation is required, or operation must be carried out in an atmosphere substituted by nitrogen or light shaded environment. Otherwise, an error may be given to measurement.
Methods using an electron carrier (mediator) include one in which an analyte is measured with high sensitivity by carrying out an enzyme reaction for a predetermined time to oxidize/reduce the electron carrier during that time, thereby accumulating the oxidized/reduced form of the electron carrier, and reducing/oxidizing the accumulated oxidized/reduced form of the electron carrier with an electrode after the predetermined time to produce great electrochemical response. Conventionally, the accumulated oxidized/reduced form of the electron carrier has been subjected to a decomposition reaction such as reduction/oxidation by a reducing substance or oxidizing substance which is coexistent with the accumulated oxidized/reduced form of the electron carrier, whereby an error may be given to measurement.
When the detectable substance is stable without being decomposed, the quantitative relationship is ensured at the time of measurement and a more excellent S/B ratio (signal-to-background ratio) can be obtained by carrying out time integration, whereby the accuracy of analysis can be improved and sensitivity can be increased. Therefore, to develop a reaction system which forms a detectable substance which is stable and can be measured easily, many efforts have been made so far. Various reagents which have been developed so far as reaction substances which form such a stable detectable substance are listed in many handbooks, Bunseki Kagaku Binran, for example.
However, the research of a reaction system which forms such a stable substance takes much time and labor, and efforts are still being made to search for a reaction system which forms a detectable substance which is always stable and can be measured easily. Therefore, even in currently used measurement methods, there are many cases where an instable substance which is decomposed by pH, moisture content, coexistent substance such as an oxidizing/reducing substance, light or the like must be measured as the detectable substance.
An analytical testing piece, used to examine and analyze a component contained in a liquid sample such as urine, for measuring an analyte by measuring a detectable substance such as a formed dyestuff based on the chemical reaction of the analyte contained in a sample, generally comprises a test portion which is a functional portion for carrying out a series of analytical processes such as the absorption, diffusion, reaction, detection and the like of the liquid sample and a support portion for supporting the test portion, and further has a sensor, sample solution suction apparatus and the like as required. The above test portion comprises layers or areas for carrying out various functions. Generally speaking, the test portion comprises a sample suction portion for sucking the sample and introducing it thereinto; a diffusion and infiltration portion for diffusing and infiltrating the sample uniformly in the test portion; a reagent portion containing a reagent which reacts with the analyte contained in the sample; a reaction portion where a reaction such as a detection reaction occurs; a developing portion for separating a component contained in the sample, a dyestuff formed by the detection reaction or the like by a chromatography-like function such as adsorption or distribution; a time control portion for adjusting the proceeding of a reaction making use of a time during which the sample moves; a holding portion for trapping or removing a component contained in the sample, formed dyestuff or the like by an adsorption function; a detection portion for detecting a dyestuff or the like by reflectance, transmission/absorption or fluorescence; an absorbing portion for absorbing excess of a sample solution, added washing solution and developing solution to prevent a back flow, and the like.
In an actual testing piece, these portions having the above functions are not always existent independently. For example, like litmus paper in which the detection portion is the same as the sample suction portion, the reagent portion and the reaction portion, there is a case where one portion has multiple functions.
For example, there are single-layered and multi-layered testing pieces which comprise a diffusion layer which also serves as a sample suction layer, a detection layer which also serves as a reagent layer and a reaction layer, or comprise a detection layer independent from a reaction layer which also serves as a reagent layer. Most of them are bonded to a base by an adhesive layer. There is a testing piece which has a developing layer or a holding layer having a function to remove an interfering component between a reaction layer and a detection layer. There is also a testing piece in which a diffusion layer also serves as a developing layer-and is in contact with a reagent layer by an adhesive layer. When detection is carried out by measuring reflectance, a reflection layer may be formed before or after a detection layer. The sample is dropped onto the diffusion layer which also serves as the sample suction layer and diffused uniformly to dissolve a reagent contained in the reagent layer, whereby a reaction proceeds. Thus, for example, a dyestuff is produced from a dyestuff precursor. When the reagent layer and the reaction layer also serve as the detection layer, the dyestuff is directly measured. However, when an independent detection layer is provided, the produced dyestuff or the like further infiltrates and moves into the detection layer and is measured at that point (see H. G. Curme, et al., Clinical Chemistry, 24 (8), 1335–1342 (1978); B. Walter, Analytical Chemistry, 55 (4), 498A (1983); Asaji Kondo, Bunseki, 1984 (7), 534; Asaji Kondo, Bunseki, 1986 (6), 387; Bunseki Kagaku Binran, p. 8 (edited by the Japan Society for Analytical Chemistry: fourth revised edition, Maruzen (1991); and Japanese Patent Application Laid-open No. 6-213886 (1994) (Masao Kitajima et al.)).
There is also a testing piece which comprises an infiltration portion of a developing solution at an end of the testing piece on a small piece of filter paper; a sample suction portion adjacent to the infiltration portion; a reaction portion which also serves as a reagent portion (having-an enzyme immobilized thereto) near the center of the testing piece; and a detection portion which also serves as a reagent portion (having a dyestuff precursor or the like immobilized thereto), a reaction portion and a holding portion after the reaction portion and makes use of the plane movement of the sample or the like. In this case, after the sample is dropped onto the sample suction portion, the developing solution is infiltrated from the end of the testing piece to move the sample by a capillary action, the sample reacts with the enzyme in the reaction portion which also serves as the first reagent portion (having the enzyme immobilized thereto) to produce hydrogen peroxide which is then moved by the developing solution to color the dyestuff precursor or the like in the detection portion which also serves as the second reagent portion (having the dyestuff precursor or the like immobilized thereto), the reaction portion and the holding portion, and adsorb and hold the produced dyestuff or the like (detectable substance). Since the hydrogen peroxide moves along with the movement of the developing solution and a coloration reaction occurs along with the movement, when the amount of the analyte increases, the length of coloration expands, whereby the substance can be measured. (see M. P. Allen, et al., Clinical Chemistry, 36 (9), 1591–1597 (1990); D. Noble, Analytical Chemistry, 65 (23), 1037A (1993).)
This testing piece is used in a urine test, a biochemical test, an immunochromatography test and the like. In an example of a testing piece for immunochromatography, when one end of filter paper having an antibody immobilized thereto (it can be said that the entire surface thereof serves as a reagent portion, a reaction portion, a developing portion, a holding portion and a detection portion) is immersed in a developing solution prepared by mixing a sample containing an antigen (analyte) and an enzyme-linked antigen as a reagent to develop with a color developing solution which is a second reagent (containing a dyestuff precursor), a portion containing the enzyme-linked antigen which has been developed and captured is colored like a belt. The length of the colored belt is proportional to the amount of the antigen contained in the sample. (see R. F. Zuk, et al., Clinical Chemistry, 31 (7), 1144–1150 (1985).)
As another example of a testing piece for immunochromatography, there is a testing piece which comprises a reagent portion (first antibody immobilized colored latex) which also serves as a sample suction portion at one end on a small piece of a membrane filter, a reagent portion (second antibody which recognizes the same antigen as that of the first antibody but is different in epitope) which also serves as a developing portion near the center, a developing portion and further a detection portion which also serves as a reagent portion (anti-first antibody antibody) and a holding portion. When a sample is dropped onto the sample suction portion, an antigen-antibody reaction between an antigen (analyte) and the first antibody occurs, an immuno-complex directly moves along with the movement of the sample, and a sandwich reaction between the immuno-complex and the second antibody occurs in the reagent portion which also serves as a developing portion. However, excess of the first antibody which does not form an immuno-complex passes through the developing portion along with the movement of the sample and is captured in the detection portion which also serves as the reagent portion, (anti-first antibody antibody) and the holding portion. The analyte can be measured by measuring the coloration of the colored latex (containing a dyestuff as a detectable substance) to which the first antibody is immobilized. (see I. W. Davidson, Analytical Proceedings, 29, 459 (1992).)
However, in the above testing pieces, a dyestuff or the like produced by a reaction with a component to be analyzed has solubility in a sample solution, reaction solution or the like in many cases with the result of such inconvenience as the elution of the dyestuff or the like into a bulk solution, a back flush to the diffusion layer, and the adhesion of the dyestuff or the like to the adjacent test portion in multi-item test paper having a plurality of test portions. Due to the movement of the dyestuff or the like toward the edge of the test portion by drying, there occurs such a phenomenon that the concentration of a center portion becomes low and that of a peripheral portion becomes high.
Such an inconvenient phenomenon that deteriorates measurement sensitivity, precision and accuracy is particularly marked in urine test paper or the like which is immersed in a sample solution for measurement but is very common irrespective of the type of sample.
Meanwhile, there have been proposed a method for preventing the elution of a reagent by covering a test portion (Japanese Patent Application Laid-open No. 2-38861 (1990)), a method for preventing liquid junction between adjacent test portions by causing the test portions composed of a porous structure (such as a porous layer or a porous film) having high absorptivity to uniformly absorb a sample (Japanese Patent Application Laid-open No. 2-6541 (1990)), a method for selecting a reaction for forming an insoluble dyestuff, a method for capturing a formed dyestuff using an insoluble and hydrophobic binder (fixing agent) (Japanese Patent Application Laid-open No. 7-181174 (1995)), a method for increasing the distance between adjacent test portions in the multi-item test paper, a method for controlling and adjusting immersion time, a method for controlling time so that measurement is carried out before diffusion, and the like. However, covering a test portion or preparing a porous structure by a precipitation-solidification method makes a test paper production process complicated. When a reaction for forming an insoluble dyestuff is selected, a product inhibition of enzyme activity occurs. A testing piece prepared by using a hydrophobic polymer as a binder has such a defect that the absorptivity of an aqueous sample solution deteriorates. A multi-item testing piece has such a defect that when the distance between adjacent test portions is increased, a larger area is required or it is disadvantageous for the movement of the sensor as a single sensor moves through a plurality of test portions to measure reflected light. The other methods have respective problems to be solved. For example, the method for controlling immersion time is troublesome in an urine test, the method for controlling time is not easy because of the relationship between control time and reaction time. Satisfactory solutions to these problems are yet to be found.
A method for measuring an analyte from electrochemical response at the time of oxidation-reduction using the above electron carrier (mediator), a method for measuring ions as an analyte by measuring the potential of a membrane upon the movement of a complex compound formed by using a ligand (ionophore) which is coordinately bonded or ion bonded to a specific ion in a liquid film electrode, and the like are known as important measuring methods. Generally speaking, in an electrode composed of an oxidized/reduced form of an electron carrier or a complex compound, the elution or diffusion of the electron carrier or ligand is prevented by adding the electron carrier or ligand to an insoluble polymer, and the electron carrier or ligand is held near the surface of the electrode so that electrons can move quickly at the same time. Since the movement of a substance in a polymer is limited, a reaction between an analyte contained in the sample or an intermediate substance produced from the analyte and the electron carrier or ligand contained in the insoluble polymer is interrupted. A satisfactory solution to this fundamental problem is yet to be found as well.